When platelets are passed through columns of thrombin covalently-attached to Sepharose at 4 degress, they elute without secreting their dense granules. However, they have been stimulated as seen by the fact that they will undergo secretion in the absence of thrombin when warmed to 37 degrees. The platelets eluting at 4 degrees are termed "cold posed". Thus we are able to determine which biochemical events have already taken place following the stimulation but prior to the release reaction. We have already demonstrated that there is an increase in the extent of phosphorylation of five proteins (81K, 56K, 36K, 27K, and 20K daltons) in the cold-poised platelet comparable to that occurring at 37 degrees. There is also a decrease in phosphorylation in one protein (67K). The specific activities of labeled Pi and ATP were equivalent following platelet stimulation. This demonstrated that the increase in phosphorylation was the result of either increased phosphorylase or decreased phosphatase activity and that the alteration of activity of the effecting enzyme also takes place at low temperatures following platelet stimulation. We are currently determining the effects of various platelet inhibitors on the secretion of cold-poised platelets when rewarmed and on protein phosphorylation. We are also determining the synthesis of prostaglandins in the cold-poised platelet. Preliminary results have shown that when cold-poised platelets are rewarmed, there is no evidence of arachidonate formation or thromboxane B2 synthesis. On the other hand, it has been shown that platelet phospholipase A2 was not inactivated during stimulation in the cold. This phenomenon is being studied further.